Morphological and molecular identification of a lung fluke, Paragonimus macrorchis (Trematoda, Paragonimidae), found in central Lao PDR and its molecular phylogenetic status in the genus Paragonimus

•Pargonimus macrorchis was found in the mountain crabs in Lao PDR.•Adult worms were obtained by experimental infection in Mongolian gerbils.•P. macrorchis is a morphologically and genetically distinct species.•Phylogenetic analyses revealed that P. macrorchis forms an independent cluster.•Methods for molecular identification and classification of the species are presented.

Paragonimus macrorchis is rather a rare species with sporadic discovery reports. To date, little is known about morphological features and the molecular phylogenetic status of P. macrorchis. Here we provide such information on P. macrorchis, of which metacercariae were collected from freshwater crabs in Khammouane Province, central Lao PDR. After morphological observation, metacercariae were excysted and were injected intra-peritoneally into Mongolian gerbils. Paragonimus adult worms were collected from the lungs of experimental gerbils 45 days after infection. A small piece of body tissue was cut at the posterior part of each adult worm for genomic DNA extraction. Then, the adult worms were stained and mounted for morphological identification. The second internal transcribed spacer region (ITS2) of rDNA and partial cytochrome c oxidase subunit 1 (cox1) gene were amplified using PCR method and sequenced. The results of morphological identification of metacercariae and adult worms together with their DNA sequences of ITS2 and partial cox1 gene clearly show that the specimens we collected in the central Lao PDR were P. macrorchis. Phylogenetic analyses revealed that P. macrorchis forms an independent cluster from other Paragonimus species in Asia.

Graphical abstractAn adult worm of Paragonimus macrorchis recovered from gerbil lung 45 days postinfection with excysted metacercariae. An adult worm was stained with carmine staining (a), drawing diagram of adult worm (b). Scale bar = 1 mm.Figure optionsDownload full-size imageDownload as PowerPoint slide